Human noroviruses (HuNoVs) are the major cause of non-bacterial gastroenteritis worldwide, resulting in significant mortality, morbidity and economic cost. However, no effective vaccines or antivirals exist for HuNoV. This is in large part due to the inability to culture HuNoV in the laboratory. This lack of a HuNoV cell culture model has prevented scientists from studying fundamental aspects of HuNoV biology, identifying therapeutic targets, and it has stifled the ability to rapidly identify antiviral drug candidates. hus, there is a critical need to develop a reproducible tissue culture system for HuNoV. The long-term goal is to elucidate mechanisms of HuNoV-host interactions and develop effective HuNoV disease prevention and control strategies. The objective of this project is to determine a cell type that reproducibly supports HuNoV infection in culture. The central hypothesis is that HuNoVs infect intestinal immune cells alone or in co-culture with intestinal epithelial cells. This hypothesis is based on our data in a newly developed HuNoV mouse model demonstrating that HuNoV infects macrophages. In addition, work by others shows that HuNoV infects B cells and dendritic cells in the intestinal lamina propria of chimpanzees, HuNoV virus-like particles bind to intestinal lamina propria cells, and tissue sections of human duodenal biopsy specimens stain positive for HuNoV antigen. It has long been presumed that HuNoV infects intestinal epithelial cells, but infection of intestinal immune cells would provide an explanation for the failed attempt to culture HuNoV in epithelial cells. The hypothesis will be tested by pursuing two specific aims: 1) Determine whether HuNoV replicates in human intestinal immune cells, and 2) Determine whether HuNoV replicates in immune cells following co-culture with 3D primary human intestinal mini-organs. In the first aim, we will directly infect intestinal immune cells from human small intestinal tissue, macrophages or dendritic cells derived from human pluripotent stem cells and established human immune cell lines. In the second aim, primary and stem cell-derived immune cells will first be co-cultured with human intestinal mini-organs prior to infection. This approach is innovative conceptually because the research tests a paradigm-shifting hypothesis, i.e., that HuNoVs replicate in immune cells instead of enterocytes in the intestine. In addition, the development of primary human immune cell/epithelial co-cultures represents a technical innovation of the human intestinal mini-organ system and will provide a new model for studying enteric infections. The proposed research is further significant because it is expected to result i the development of a reproducible tissue culture model for HuNoVs. Such a development would overcome a major barrier in the field, which has hampered HuNoV research for the past 40 years. Ultimately, such technical break-through will provide the basis for future advances in HuNoV research both at the basic and translational level.